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Oxidative stress induced mitochondrial protein kinase A mediates cytochrome c oxidase dysfunction.

Srinivasan S, Spear J, Chandran K, Joseph J, Kalyanaraman B, Avadhani NG - PLoS ONE (2013)

Bottom Line: Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS).Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production.The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology and the Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.

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Effects of antioxidants on mitochondrial PKAα subunit level and activity.RAW 264.7 macrophages were subjected to hypoxia for 12h with or without addition of 1µM Mito-CP or 10mM N-Acetyl Cysteine. At the end of hypoxia, part of the cells was used for measuring ROS production by DCFDA oxidation and the remaining for mitochondria isolation. Protein was estimated by Lowry’s method. A) PKA activity and ROS production (n=3). After hypoxia cells were plated in 96 well plate in phosphate buffered saline and incubated with DCFDA (1µM) for 15 minutes. Fluorescence was measured at Excitation 525nm and Emission 575nm. Corresponding PKA activity was measured in 10µg of mitochondrial protein, B) PKAα protein level. 30µg of mitochondrial protein was separated on SDS PAGE and transferred to nitrocellulose membrane. PKAα and CcO IVi1 antibodies were used for immunoblotting. Relative band intensities are given in parantheses. The blot is representative of two separate experiments. C) Effect of Mito-CP on CcO activity under hypoxia. CcO activity was measured with 10µg of mitochondria (n=4). **, p<0.005.
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pone-0077129-g003: Effects of antioxidants on mitochondrial PKAα subunit level and activity.RAW 264.7 macrophages were subjected to hypoxia for 12h with or without addition of 1µM Mito-CP or 10mM N-Acetyl Cysteine. At the end of hypoxia, part of the cells was used for measuring ROS production by DCFDA oxidation and the remaining for mitochondria isolation. Protein was estimated by Lowry’s method. A) PKA activity and ROS production (n=3). After hypoxia cells were plated in 96 well plate in phosphate buffered saline and incubated with DCFDA (1µM) for 15 minutes. Fluorescence was measured at Excitation 525nm and Emission 575nm. Corresponding PKA activity was measured in 10µg of mitochondrial protein, B) PKAα protein level. 30µg of mitochondrial protein was separated on SDS PAGE and transferred to nitrocellulose membrane. PKAα and CcO IVi1 antibodies were used for immunoblotting. Relative band intensities are given in parantheses. The blot is representative of two separate experiments. C) Effect of Mito-CP on CcO activity under hypoxia. CcO activity was measured with 10µg of mitochondria (n=4). **, p<0.005.

Mentions: Since hypoxia did not result in any increase in cAMP level either in cytosol or mitochondria and activation of PKA was unaffected by adenylate cyclase inhibitor, KH7, we investigated alternative mechanisms of activation of PKA. Oxidative stress and ROS has been implicated in the cAMP independent activation of PKA [42–44]. We therefore tested the effects of two different antioxidants on hypoxia modulated mitochondrial PKA activation. Figure 3A shows that both general antioxidant N-Acetyl Cysteine and mitochondria targeted Mito-CP not only reduced ROS levels, but also attenuated mitochondrial PKA activation in cells subjected to hypoxia. Treatment with the targeting vehicle TPP+ alone did not affect hypoxia mediated increases in ROS levels as well as mitochondrial PKA activity. Immunoblot in Figure 3B shows that the antioxidants also attenuated hypoxia mediated increase in the level of mitochondrial PKAα subunit. Decreased mitochondrial PKA activation was accompanied by significant protection of CcO activity under hypoxia (Figure 3C).These results suggest that activation of PKA under hypoxia is associated with increased ROS production. Inhibition of ROS and prevention of loss of CcO activity by mitochondria targeted antioxidant suggests that the activation is predominantly, if not exclusively because of mitochondrially generated ROS.


Oxidative stress induced mitochondrial protein kinase A mediates cytochrome c oxidase dysfunction.

Srinivasan S, Spear J, Chandran K, Joseph J, Kalyanaraman B, Avadhani NG - PLoS ONE (2013)

Effects of antioxidants on mitochondrial PKAα subunit level and activity.RAW 264.7 macrophages were subjected to hypoxia for 12h with or without addition of 1µM Mito-CP or 10mM N-Acetyl Cysteine. At the end of hypoxia, part of the cells was used for measuring ROS production by DCFDA oxidation and the remaining for mitochondria isolation. Protein was estimated by Lowry’s method. A) PKA activity and ROS production (n=3). After hypoxia cells were plated in 96 well plate in phosphate buffered saline and incubated with DCFDA (1µM) for 15 minutes. Fluorescence was measured at Excitation 525nm and Emission 575nm. Corresponding PKA activity was measured in 10µg of mitochondrial protein, B) PKAα protein level. 30µg of mitochondrial protein was separated on SDS PAGE and transferred to nitrocellulose membrane. PKAα and CcO IVi1 antibodies were used for immunoblotting. Relative band intensities are given in parantheses. The blot is representative of two separate experiments. C) Effect of Mito-CP on CcO activity under hypoxia. CcO activity was measured with 10µg of mitochondria (n=4). **, p<0.005.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3795003&req=5

pone-0077129-g003: Effects of antioxidants on mitochondrial PKAα subunit level and activity.RAW 264.7 macrophages were subjected to hypoxia for 12h with or without addition of 1µM Mito-CP or 10mM N-Acetyl Cysteine. At the end of hypoxia, part of the cells was used for measuring ROS production by DCFDA oxidation and the remaining for mitochondria isolation. Protein was estimated by Lowry’s method. A) PKA activity and ROS production (n=3). After hypoxia cells were plated in 96 well plate in phosphate buffered saline and incubated with DCFDA (1µM) for 15 minutes. Fluorescence was measured at Excitation 525nm and Emission 575nm. Corresponding PKA activity was measured in 10µg of mitochondrial protein, B) PKAα protein level. 30µg of mitochondrial protein was separated on SDS PAGE and transferred to nitrocellulose membrane. PKAα and CcO IVi1 antibodies were used for immunoblotting. Relative band intensities are given in parantheses. The blot is representative of two separate experiments. C) Effect of Mito-CP on CcO activity under hypoxia. CcO activity was measured with 10µg of mitochondria (n=4). **, p<0.005.
Mentions: Since hypoxia did not result in any increase in cAMP level either in cytosol or mitochondria and activation of PKA was unaffected by adenylate cyclase inhibitor, KH7, we investigated alternative mechanisms of activation of PKA. Oxidative stress and ROS has been implicated in the cAMP independent activation of PKA [42–44]. We therefore tested the effects of two different antioxidants on hypoxia modulated mitochondrial PKA activation. Figure 3A shows that both general antioxidant N-Acetyl Cysteine and mitochondria targeted Mito-CP not only reduced ROS levels, but also attenuated mitochondrial PKA activation in cells subjected to hypoxia. Treatment with the targeting vehicle TPP+ alone did not affect hypoxia mediated increases in ROS levels as well as mitochondrial PKA activity. Immunoblot in Figure 3B shows that the antioxidants also attenuated hypoxia mediated increase in the level of mitochondrial PKAα subunit. Decreased mitochondrial PKA activation was accompanied by significant protection of CcO activity under hypoxia (Figure 3C).These results suggest that activation of PKA under hypoxia is associated with increased ROS production. Inhibition of ROS and prevention of loss of CcO activity by mitochondria targeted antioxidant suggests that the activation is predominantly, if not exclusively because of mitochondrially generated ROS.

Bottom Line: Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS).Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production.The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology and the Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.

Show MeSH
Related in: MedlinePlus